It is known to make aspartame by enzymatic coupling of blocked aspartic acid with phenylalanine methyl ester. While the mechanism is undoubtedly complex, the overall result is a simple dehydration, thus: ##STR14##
The amine on aspartic acid is blocked with carbobenzoxy (i.e., a "Q" group) to prevent undesirable side reactions.
Aspartame is useful as a sweetener only in the L,L-form, i.e., when its 2 chiral carbons (in this instance one in the phenylalanine moiety and one in the aspartic acid moiety) are in the L form. The 3 other optically-active isomers (L,D; D,L; and D,D) are bitter or tasteless. Procedures using phenylalanine are known that will give the L,L-form, substantially free of undesirable isomers.
U.S. Pat. No. 4,284,721, Oyama et al, discloses the foregoing reaction to give the L,L-form, using various immobilized enzymes, including thermolysin. The pores of the immobilized enzyme matrix are filled with water, and thus the reaction of aspartic acid and phenylalanine is carried out in water. The 2 reactants are however dissolved in an organic solvent immiscible with water (e.g., ethyl acetate), and that solution contacts the water-containing immobilized enzyme. Yields of L,L-ZAPM are stated variously as 25.5-88%. The inventors in 4,284,721 published a parallel paper dealing with the same reaction, reactants, and enzyme, Oyama et al, J. Org. Chem., 1981, 46, 5241-5242, stating, " . . . substrates move from the organic layer to the aqueous layer of the support, where the reaction takes place, and then the product diffuses back to the organic layer effectively . . . " This paper also mentions that in organic solvents "the reaction rate is rather slow as compared with that in aqueous solution." And see Oyama et al, Enzymatic Production of Aspartame, Enzyme Engineering, 7, pp. 96-98, disclosing reaction of L-aspartic acid with D,L-phenylalanine to give L,L-aspartame, using thermolysin. The reaction is carried out in water. The reaction product is in the form of an "insoluble addition compound", ZAPM.PheOMe. (See Isowa below.) The Z group is removed by catalytic hydrogenation.
Isowa et al, Tetrahedron Letters, No. 28, pp. 2611-2612 (1979), discloses that the thermolysin-induced reaction of Z-L-aspartic acid with L-phenylalanine-OMe in water gives Z-L-Asp-L-Phe-OMe-L-Phe-Ome; which is to say, the L,L-reaction product forms an addition product with the PheOMe reactnat. The enzyme was not immobilized. When racemic mixes of reactants were used, only the L,L-apartame product was precipitated as the addition compound. The phenylalanine portion was separated by use of aqueous hydrochloric acid and the Z group removed by catalytic hydrogenolysis, thereby to give free L,L-aspartame. Yields are high, typically in excess of 90%. Formation of such addition compounds by enzymatic coupling is aqueous media is also described in U.S. Pat. Nos. 4,116,768, 4,119,493, 1,165,311, 4,256,836, and 4,436,925.
Petkov et al, Enzyme Peptide Synthesis, Tetrahedron Letters, 25, No. 34, pp. 3751-3754 (1984) teaches reaction of Z-Asp with PheOMe in water using thermolysin. With excess PheOMe an addition compound is formed (per Isowa et al supra). Reaction times of 3-4 hours give excellent yields (typically in excess of 90%).
To summarize certain of the prior art, the reaction of Z-aspartic acid with phenylalanine methyl ester, using immobilized thermolysin:
(a) in water, the reaction is fast, with good yield of an addition compound, Z-L,L-Asp.PheOMe;
(b) in organic solution, the reaction is slower, but no addition compound separates;
(c) whether in water or organic solution, thermolysin forms L,L-aspartame from racemic reactants, i.e., L,D-Phe+L,D-Asp.
In one step of the instant invention methyl 2-oximino benzoylacetate is hydrogenated to make erythro-beta-phenylserine methyl ester. See Example 4. In that connection the following articles is of interest.
Elphimoff-Felkin et al, Memoires Presentes a La Societe Chimique (1952), pp. 252-264, at p. 259, disclose hydrogenation of ethyl 2-oximine benzoyl acetate, dissolved in acetic acid, in the presence of PtO.sub.2, using hydrogen. Thye report a mix of threo and erythro isomers of phenylserine, stating that the erythro isomer predominated. A repetition of their work confirms their result, the mix analyzing 75% erythro isomer and 25% threo isomer. The corresponding reaction in the instant invention differs in use of catalyst (Pd metal, not the French PtO.sub.2) and in the use of solvent (methanol, not the French acetic acid). These differences result in a yield of essentially pure erythro isomer, and such result ws not be predicted. Using the reference French procedure, 1 g of oxime gives 600 mg of erythro isomer and 200 mg of threo isomer, an overall yield of 92% (based on oxime) and an erythro isomer yield of 54.3%, based on oxime. This compares with yields of 95+% of pure erythro isomer obtained in the invention process, same basis. See Example 4, using methyl ester, and Example 5, using ethyl ester.